Tenocytic induction of stem cells from bone marrow on polymeric microparticles for a new concept of tendon regenerative prosthesis

A new concept of a regenerative and resorbable prosthesis for tendon and ligament has been developed. The prosthesis consists of a poly-lactide acid (PLA) braid, microparticles in its interior serving as cell carriers, and a surface non-adherent coating. The aim of this study is to select the most suitable support, microparticles of poly-L-lactide (PLLA) or chitosan (CHT), for carrying the cells inside the hollow PLA braid. Microparticles of these polymers were manufactured and blended with microparticles of hyaluronic acid (HA). All of them were physically and biologically characterized. Cell viability, morphology and proliferation of human mesenchymal stem cells (hMSCs) on the different supports were evaluated and compared, revealing that PLLA microparticles were the most appropriate to be used as injectable cell-carrier. Finally, hMSCs differentiation into tenocytes was carried out on PLLA microparticles using bone morphogenetic protein-12 (BMP-12) and a mixture of transforming growth factor-β1 (TGF-β1) and insulin-like growth factor1 (IGF-1). Cell morphology was analyzed by electronic and confocal microscopy and cell differentiation was evaluated immunocytochemically for the presence of type I collagen and tenomodulin. Besides, the tenomodulin and decorin gene expression were measured by real-time quantitative polymerase chain reaction (RT-qPCR). Our results showed that the medium supplemented with BMP-12 promoted higher expression of tenomodulin and decorin, both of them differentiation markers of tenocytes. This approach might be relevant to future tissue engineering applications in reconstruction of tendon and ligament defects. Authors acknowledge support of the Spanish Instituto de Salud Carlos III through CIBERbbn and the Spanish Network on Cell Therapy (Red TerCel) initiatives.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Service-learning educational approach for undergraduate students: development of an outreach workshop for high school students

In the last decade, research institutes and universities have strengthened the development of outreach activities in the biomedical field, involving researchers and professors as well as graduate students, but with little or no implication of undergraduate students. However, the development of this type of activities, using the Service-Learning educational approach, could be a valuable tool that would manage the acquisition of learning competencies by undergraduate students of Health Science Degrees and would put science at the service of society. In this project, we present the development of the workshop entitled “Exploring the human body”, in which 205 students in their first and second year of a Degree in Nursing or Medicine (University of Málaga, Spain) acted as mentors of 753 high school students (15 to 16 years old) in several school years (since 2016-2017, excluding 2019-2020 and 2020-2021 due to the COVID-19 pandemic). The workshop consisted of five work stations. Each station featured a set of different experiments and activities that were designed to teach the multiple levels by which the human body, and particularly the nervous system, can be studied: biomolecules, cells, tissues, organs and systems. Both high school and undergraduate students gave an evaluation of the workshop via questionnaires (Likert scale-based and short-answer questions) and a debriefing with the university professors. Data showed an overall score of 4.6 out of 5 points for the workshop by both high school and undergraduate students. In addition, undergraduate students pointed out that their participation had a positive impact on their academic background (4.8 out of 5 points), mainly due to the improvement of their oral communication skills (78 students) and self-confidence (58 students).Universidad de Málaga. Servicio de Publicaciones y Divulgación Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Antioxidant and neuroprotective actions of IGF-II against glucocorticoid-induced toxicity in dopaminergic neurons.

The neurodegenerative Parkinson’s disease (PD) affects 1–3% of the population aged over 65. A wide range of pathways and mechanisms are involved in its pathogenesis, such as oxidative stress, mitochondrial dysfunction, inflammation and neuronal glucocorticoid-induced toxicity, which ultimately produce a progressive loss of nigral dopamine neurons. Insulin-like growth factor II (IGF-II) has shown antioxidant and neuroprotective effects in some neurodegenerative disorders. Therefore, our aim was to study IGF-II protective effects against oxidative damage on a cellular combined model of PD and mild to moderate stress, based on corticosterone (CORT), an endocrine response marker to stress, and the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+). The dopaminergic neuronal cell line SN4741 (RRID:CVCL_S466) derived from mouse substantia nigra were exposed to 200 μM MPP+, 0.5 μM CORT or both, with or without 25 ng/mL IGF-II, for 2.5 or 6 h. Cell viability, oxidative stress parameters, mitochondrial and dopamine markers and intracellular signaling pathways were evaluated. The administration of MPP+ or CORT individually led to cell damage compared to control situations, whereas the combination of both drugs produced very considerable toxic synergistic effect. IGF-II counteracts the mitochondrial-oxidative damage, protecting dopaminergic neurons from death and neurodegeneration. IGF-II maintained the tyrosine hydroxylase expression and promotes nuclear factor (erythroid-derived 2)-like 2 antioxidant response in a glucocorticoid receptor-dependent pathway, preventing oxidative cell damage and maintaining mitochondrial function. This work revealed the potential neuroprotective role of IGF-II to protect nigral dopamine neurons against mitochondrial-oxidative damage induced by CORT and MPP+ was demonstrated. Thus, IGF-II is a potential therapeutic tool for prevention and treatment of PD patients suffering mild to moderate emotional stress.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Insulin-like growth factor II neuroprotective effects against mitochondrial-oxidative and neuronal damage induced by CORT and MPP+ in dopaminergic neurons

Aims: Parkinson’s disease (PD) affects 1–3% of the population aged over 65. Stress seems to contribute to PD neuropathology, probably by dysregulation of the hypothalamic–pituitary–adrenal axis. Key factors are oxidative stress, mitochondrial dysfunction and neuronal glucocorticoid-induced toxicity. Insulin-like growth factor II (IGF-II) has shown antioxidant and neuroprotective effects in some neurodegenerative disorders. Therefore, our aim was to study IGF-II protective effects against oxidative damage on a cellular combined model of PD and mild to moderate stress, based on corticosterone (CORT) and the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+). Methods: The dopaminergic neuronal cell line SN4741 (RRID:CVCL_S466) derived from mouse substantia nigra were exposed to 200 μM MPP+, 0.5 μM CORT or both, with or without 25 ng/mL IGF-II, for 2.5 or 6 h. Cell viability, oxidative stress parameters, mitochondrial and dopamine markers and intracellular signaling pathways were evaluated. Results: The administration of MPP+ or CORT individually led to cell damage compared to control situations, whereas the combination of both drugs produced very considerable toxic synergistic effect. IGF-II counteracts the mitochondrial-oxidative damage, protecting dopaminergic neurons from death and neurodegeneration. IGF-II promotes PKC activation and nuclear factor (erythroid-derived 2)-like 2 antioxidant response in a glucocorticoid receptor-dependent pathway, preventing oxidative cell damage and maintaining mitochondrial function. Conclusions: IGF-II capacity to protect nigral dopamine neurons against mitochondrial-oxidative damage induced by CORT and MPP+ was demonstrated. Thus, IGF-II is a potential therapeutic tool for prevention and treatment of PD patients suffering mild to moderate emotional stress.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

IGF-II treatment prevents the oxidative damage derived by MPP+/MPTP administration in a cellular and animal model of Parkinson’s disease.

El factor de crecimiento de la insulina-II (IGF-II) ha mostrado efectos antioxidantes y neuroprotectores en algunos trastornos neurodegenerativos, como es la Enfermedad de Parkinson (EP). Analizamos el efecto de IGF-II y la implicación de la esfingosina kinasa (SPHK), en la citoarquitectura/función mitocondrial tras provocar daño oxidativo con la administración de la neurotoxina MPTP y su metabolito activo. La línea celular SN4741 se trató con MPP+ solo/en presencia de IGF-II (2 h). Los tratamientos fueron reemplazados por medio/IGF-II (2h), respectivamente. Se estudió: morfología (microscopía electrónica, EM), tasa de consumo de oxígeno (OCR) y muerte celular (LDH). MPTP/probenecid(p) por vía parenteral (35d) indujo un daño progresivo en la vía nigroestriatal dopaminérgica (DA-NSP) de los animales. En los días 36-44, se inyectó vehículo. El grupo control recibió vehículo siguiendo el mismo régimen de administración (1-44d). Otro grupo se trató con IGF-II una vez inducido el daño por MPTP (22-44 días). Para investigar el papel de IGF-II en la alteración conductual inducida por MPTP/p, se evaluó el rendimiento motor. Se realizó inmunotinción para marcadores dopaminérgicos y astrogliosis. Las células tratadas con MPP+ mostraron menos mitocondrias (EM) y con pérdida parcial/total de crestas, alteraciones de membrana y forma hinchada. IGF-II mantuvo el número de mitocondrias con morfología similar al control. La disminución de OCR tras la administración de MPP+ (30%) se recuperó con IGF-II. SPHK está implicada en este mecanismo, como indica su inhibición (MPP++IGF-II+MPA-08 6 veces > LDH vs MPP++IGF-II). En animales, IGF-II recuperó el efecto de MPTP sobre los marcadores DA-NSP y sobre el rendimiento motor. IGF-II contrarresta el aumento del estrés oxidativo y la disfunción mitocondrial inducida por la neurotoxina, el deterioro conductual y la degeneración de DA-NSP. SPHK estaría involucrada en este mecanismo.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Neuronal Metabolism and Neuroprotection: Neuroprotective Effect of Fingolimod on Menadione-Induced Mitochondrial Damage

Imbalance in the oxidative status in neurons, along with mitochondrial damage, are common characteristics in some neurodegenerative diseases. The maintenance in energy production is crucial to face and recover from oxidative damage, and the preservation of different sources of energy production is essential to preserve neuronal function. Fingolimod phosphate is a drug with neuroprotective and antioxidant actions, used in the treatment of multiple sclerosis. This work was performed in a model of oxidative damage on neuronal cell cultures exposed to menadione in the presence or absence of fingolimod phosphate. We studied the mitochondrial function, antioxidant enzymes, protein nitrosylation, and several pathways related with glucose metabolism and glycolytic and pentose phosphate in neuronal cells cultures. Our results showed that menadione produces a decrease in mitochondrial function, an imbalance in antioxidant enzymes, and an increase in nitrosylated proteins with a decrease in glycolysis and glucose-6-phosphate dehydrogenase. All these effects were counteracted when fingolimod phosphate was present in the incubation media. These effects were mediated, at least in part, by the interaction of this drug with its specific S1P receptors. These actions would make this drug a potential tool in the treatment of neurodegenerative processes, either to slow progression or alleviate symptoms.This research was supported by the following projects: PS13/14: Study of the non immunological mechanisms of action of Gilenya (Fingolimod) as therapeutic tool in Multiple Sclerosis and/or other neurodegenerative diseases, Novartis Farmacéutica S.A.; CTS507 and CTS156 from Consejería de Economía Innovación Ciencia y Empresa, Junta de Andalucía. N. Valverde was supported by Proyectos I+D+I-Programa Operativo FEDER Andalucía 2014-2020 (UMA18-FEDERJA 004) Junta de Andalucía and Fondo Social Europeo (EU).Ye

Impact of Glucocorticoid on a Cellular Model of Parkinson’s Disease: Oxidative Stress and Mitochondrial Function

Stress seems to contribute to the neuropathology of Parkinson’s disease (PD), possibly by dysregulation of the hypothalamic–pituitary–adrenal axis. Oxidative distress and mitochondrial dysfunction are key factors involved in the pathophysiology of PD and neuronal glucocorticoid-induced toxicity. Animal PD models have been generated to study the effects of hormonal stress, but no in vitro model has yet been developed. Our aim was to examine the impact of corticosterone (CORT) administration on a dopaminergic neuronal cell model of PD induced by the neurotoxin MPP+, as a new combined PD model based on the marker of endocrine response to stress, CORT, and oxidative-mitochondrial damage. We determined the impact of CORT, MPP+ and their co-incubation on reactive oxygen species production (O2−•), oxidative stress cellular markers (advanced-oxidation protein products and total antioxidant status), mitochondrial function (mitochondrial membrane potential and mitochondrial oxygen consumption rate) and neurodegeneration (Fluoro-Jade staining). Accordingly, the administration of MPP+ or CORT individually led to cell damage compared to controls (p < 0.05), as determined by several methods, whereas their co-incubation produced strong cell damage (p < 0.05). The combined model described here could be appropriate for investigating neuropathological hallmarks and for evaluating potential new therapeutic tools for PD patients suffering mild to moderate emotional stress